A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl- from acidic organelles in amacrine cells

摘要

gamma-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl-. However, the electrochemical gradient for Cl- across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl- levels support inhibition, whereas higher levels of cytosolic Cl- can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl- from acidic organelles and transiently elevates cytosolic Cl-, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl-. We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl- channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl-. To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl- from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl- from acidic organelles.